EP3379506A1 - Xanthurenic acid derivative pharmaceutical compositions and methods related thereto - Google Patents

Xanthurenic acid derivative pharmaceutical compositions and methods related thereto Download PDF

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Publication number
EP3379506A1
EP3379506A1 EP18158959.9A EP18158959A EP3379506A1 EP 3379506 A1 EP3379506 A1 EP 3379506A1 EP 18158959 A EP18158959 A EP 18158959A EP 3379506 A1 EP3379506 A1 EP 3379506A1
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EP
European Patent Office
Prior art keywords
glucoside
pharmaceutical composition
xanthurenic acid
use according
optionally substituted
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP18158959.9A
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German (de)
English (en)
French (fr)
Inventor
Michael Schmertzler
Mark Mitchnick
Christopher D. Cain
Stewart Shankel
Neal Bricker
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Naturon Inc
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Naturon Inc
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Publication of EP3379506A1 publication Critical patent/EP3379506A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/48—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/20—Oxygen atoms
    • C07D215/22—Oxygen atoms attached in position 2 or 4
    • C07D215/233—Oxygen atoms attached in position 2 or 4 only one oxygen atom which is attached in position 4
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    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/02—Heterocyclic radicals containing only nitrogen as ring hetero atoms
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Definitions

  • Diuretics are a group of drugs used to treat a variety of medical conditions, including congestive heart failure, hypertension, certain types of liver and kidney diseases and increased intra-ocular pressure. Diuretics act on the transport of sodium (Na + ) by the nephrons of the kidney so as to increase the renal excretion of Na + (and associated ions) and water out of the body and thereby to decrease the extracellular fluid (ECF) volume. Normally, Na + enters the ECF via the diet, and is excreted in the urine in amounts identical to the intake. In normal adults over 99% of the sodium entering the nephrons of the two kidneys (via glomerular filtration) is transported via an energy dependent process out of the tubular fluid and back into the ECF.
  • Na + sodium
  • ECF extracellular fluid
  • the principle drugs which are included in the "diuretic” category act by inhibiting the transport of Na + (and water) out of the tubular fluid by acting on a specific "carrier” in the tubular epithelial cells at a specific site of the nephron. The latter varies with the diuretic employed.
  • Loop diuretics also known as high-ceiling diuretics, act on the thick ascending loop of Henle within the kidney. Examples include furosemide, bumetanide and toresemide. Loop diuretics have a peak diuretic effect far greater than other classes of diuretics. This class acts to inhibit electrolyte reabsorption resulting in the excretion of not only sodium, but also potassium, calcium and magnesium. Loop diuretics are considered "potassium wasting.” For example, furosemide is commonly used to treat heart failure, pulmonary edema, hypertension and poisoning.
  • Thiazide-type diuretics act in the distal tubule and connecting segment of the kidneys. Examples include chlorothiazide, chlorthalidone, hydrochlorothiazide, indapamide and metolazone. Although thiazides cause less distortion of the electrolyte composition of the extra-cellular fluid than other classes of diuretics, there is also lower intensity of diuresis produced by these drugs. This class contains many sulfonamide chemical entities and thus may cause an allergic reaction in those with sulfa allergies. Although thiazides do not cause calcium excretion, potassium excretion increases with acute administration. Thiazides may also induce hyperglycemia and aggravate pre-existing diabetes mellitus. Thiazide diuretics may also cause increased serum cholesterol, low-density lipoprotein (LDL) and triglyceride concentration. Thiazides are also considered “potassium wasting" diuretics.
  • LDL low-density lipoprotein
  • Potassium-sparing diuretics may act through either of several mechanisms. Some are steroidal in structure and act in aldosterone-sensitive cells in the cortical connecting tubule in the kidney. Members in this drug class are competitive antagonists of endogenous mineralocorticoid steroids such as aldosterone, which acts to enhance sodium absorption and potassium excretion.
  • the aldosterone receptor is a soluble, cytoplasmic protein found in several tissues including salivary glands, colon and segments of nephrons in the kidney.
  • Spironolactone a representative member of this drug class, binds to the aldosterone receptor and prevents the receptor from assuming an active conformation. Spironolactone also increases calcium excretion.
  • Triamterene and amiloride are non-steroidal potassium-sparing diuretics that inhibit electrogenic entry of sodium in the late segments of the kidney nephron. Triamterene and amiloride cause an increase in sodium and chloride excretion, but have little effect on potassium excretion. Side effects of Triamterene include hyperkalemia (increased serum potassium concentration), nausea, vomiting, leg cramps and dizziness. Amiloride side effects also include hyperkalemia, nausea, vomiting, diarrhea and headache.
  • Osmotic diuretics such as mannitol
  • mannitol are poorly reabsorbed by the renal tubules. This drug class effects poor net reabsorption of sodium salts.
  • mannitol is poorly absorbed by the gastrointestinal tract, and thus must be administered intravenously.
  • Other osmotic diuretics include glycerol, urea and isosorbide.
  • Carbonic anhydrase inhibitors such as acetazolamide, cause a modest decrease of sodium reabsorption and may also cause loss of potassium and metabolic acidosis due to its mechanism of action.
  • Diuretics are used to treat high blood pressure (hypertension), either alone, or in combination with other drugs.
  • High blood pressure adds to the workload of the heart and arteries. If the condition continues for a prolonged period of time, heart and artery function may be impaired. This can damage the blood vessels of the brain, heart and kidneys, resulting in stroke, heart or kidney failure. High blood pressure may also increase the risk of heart attack. These risks can be reduced if blood pressure is properly controlled.
  • the National Heart, Lung, and Blood Institute's (NHLBI) high blood pressure guidelines ( JAMA, May 21, 2003; www.nhlbi.nih.gov ) emphasize a need to develop new diuretic medications without the side affects of the aforementioned diuretic pharmacopeia.
  • the present invention also provides methods of treating, controlling and preventing hypertension, edema, acute renal failure, congestive heart failure, chronic renal failure, ascites, increased intra-ocular pressure or nephrotic syndrome and other related diseases and conditions using pharmaceutical compositions comprising compounds of formula I.
  • aliphatic or "aliphatic group” as used herein means a straight-chain or branched C 1-12 hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic C 3-8 hydrocarbon or bicyclic C 8-12 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule wherein any individual ring in said bicyclic ring system has 3-7 members.
  • suitable alkyl groups include, but are not limited to, linear or branched or alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
  • alkoxy used alone or as part of a larger moiety include both straight and branched chains containing one to twelve carbon atoms.
  • alkenyl and alkynyl used alone or as part of a larger moiety shall include both straight and branched chains containing two to twelve carbon atoms.
  • haloalkyl means alkyl, alkenyl or alkoxy, as the case may be, substituted with one or more halogen atoms.
  • halogen or halo means F, Cl, Br or I.
  • heteroatom means nitrogen, oxygen, or sulfur and includes any oxidized form of nitrogen and sulfur, and the quaternized form of any basic nitrogen.
  • aryl used alone or in combination with other terms, refers to monocyclic, bicyclic or tricyclic carbocyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 8 ring members.
  • aryl may be used interchangeably with the term “aryl ring”.
  • aralkyl refers to an alkyl group substituted by an aryl.
  • aralkoxy refers to an alkoxy group substituted by an aryl.
  • heterocycloalkyl means monocyclic, bicyclic or tricyclic ring systems having five to fourteen ring members in which one or more ring members is a heteroatom, wherein each ring in the system contains 3 to 7 ring members and is non-aromatic.
  • heteroaryl used alone or in combination with other terms, refers to monocyclic, bicyclic and tricyclic ring systems having a total of five to fourteen ring members, and wherein: 1) at least one ring in the system is aromatic; 2) at least one ring in the system contains one or more heteroatoms; and 3) each ring in the system contains 3 to 7 ring members.
  • heteroaryl may be used interchangeably with the term “heteroaryl ring” or the term “heteroaromatic".
  • heteroaryl rings examples include 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 5-tetrazolyl, 2-triazolyl, 5-triazolyl, 2-thienyl, 3-thienyl, carbazolyl, benzimidazoly
  • An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) or heteroaryl (including heteroaralkyl, heteroarylalkoxy and the like) group may contain one or more substituents. Suitable substituents on the unsaturated carbon atom of an aryl, heteroaryl, aralkyl or heteroaralkyl group are selected from halogen; haloalkyl; -CF 3 : -R 9 ; -OR 9 ; -SR 9 ; 1,2-methylenedioxy; 1,2-ethylenedioxy; protected OH (such as acyloxy); phenyl (Ph); Ph substituted with R 9 ; -O(Ph); -O-(Ph) substituted with R 9 ; -CH 2 (Ph); -CH 2 (Ph) substituted with R 9 ; -CH 2 CH 2 (Ph); -CH 2 CH 2 (Ph) substituted with R 9 ; -NO 2 ;
  • R 9 , R 10 , R 11 or R 12 is C 1-6 aliphatic, it may be substituted with one or more substituents selected from -NH 2 , -NH(C 1-4 aliphatic), -N(C 1-4 aliphatic) 2 , -S(O)(C 1-4 aliphatic), -SO 2 (C 1-4 aliphatic), halogen, (C 1-4 aliphatic), -OH, -O-(C 1-4 aliphatic), -NO 2 , -CN, -CO 2 H, -CO 2 (C 1-4 aliphatic), -O(halo C 1-4 aliphatic), or -halo(C 1-4 aliphatic); wherein each C 1-4 aliphatic is unsubstituted.
  • R 13 or R 14 is C 1-6 aliphatic, it may be substituted with one or more substituents selected from -NH 2 , -NH(C 1-4 aliphatic), -N(C 1-4 aliphatic) 2 , halogen, -OH, -O-(C 1-4 aliphatic), -NO 2 , -CN, -CO 2 H, -CO 2 (C 1-4 aliphatic), -O(halo C 1-4 aliphatic) or -halo(C 1-4 aliphatic); wherein each C 1-4 aliphatic is unsubstituted.
  • R 15 or R 16 is a C 1-6 aliphatic group or a phenyl ring, it may be substituted with one or more substituents selected from-NH 2 , -NH(C 1-4 aliphatic), -N(C 1-4 aliphatic) 2 , halogen, -(C 1-4 aliphatic), -OH, -O-(C 1-4 aliphatic), -NO 2 , -CN, -CO 2 H, -CO 2 (C 1-4 aliphatic), -O(halo C 1-4 aliphatic) or -halo(C 1-4 aliphatic); wherein each C 1-4 aliphatic is unsubstituted.
  • saccharide defines a carbohydrate, or sugar, made up of one or more units with the empirical generic formula (CH 2 O) n .
  • a saccharide is further classified as a monosaccharide, disaccharide or polysaccharide depending on the number of units or an aminosaccharide if one or more oxygen atoms are replaced by a nitrogen atom.
  • a saccharide may also be classified as a deoxysaccharide if one or more hydroxy groups are replaced by a hydrogen atom.
  • a saccharide substituent may be further substituted on any primary or secondary hydroxy group by, for example, an alkyl, alkoxyalkyl, aryl, heteroaryl, ether, ester, acetal, carbonate or carbamate.
  • the term "monosaccharide” defines a single carbohydrate, or sugar unit.
  • Two families of monosaccharides are aldoses or ketoses. Aldoses have a carbonyl group at the end of the carbon chain as an aldehyde, when the monosaccharide is written in a linear, open-chain formula. If the carbonyl is in any other position in the carbon chain the monosaccharide is a ketone and referred to as a ketose.
  • Three carbon monosaccharides are trioses: glyceraldehydes, an aldose, and dihydroxyacetone, a ketose. Monosaccharides, except for dihydroxyacetone, have one or more asymmetric centers.
  • D- or L- refer to the configuration of the carbon atom of the chiral carbon most distant from the carbonyl carbon.
  • Monosaccharides with 4, 5, 6 and 7 carbon atoms in their backbones are termed tetroses, pentoses, hexoses, and heptoses, respectively. Each of these exists in two series: aldotetroses and ketotetroses, aldopentoses and ketopentoses, aldohexoses and ketohexoses, aldoheptoses and ketoheptoses.
  • Tetroses include erythrose and threose.
  • Pentoses include ribose, arabinose, xylose and lyxose.
  • Hexoses include allose, altrose, glucose, mannose, gulose, idose, galactose and talose.
  • Monosaccharides with 5 or more carbons in the backbone usually occur as cyclic, or ring, structures in which the carbonyl carbon has formed a covalent bond with one of the hydroxy groups along the chain.
  • Six-membered ring compounds are termed pyranoses, five-membered ring compounds are furanoses. Formation of a six-membered ring results from reaction of aldehydes and alcohols to form hemi-acetals which contain an asymmetric carbon atom.
  • One configuration around the C-1 carbon is described as ⁇ - and the other is described as the ⁇ - form.
  • disaccharide refers to a molecular moiety containing two monosaccharides covalently bound to each other. Disaccharides include maltose [glucose-glucose], lactose [galactose-glucose] and sucrose [fructose-glucose].
  • polysaccharide includes multiple monosaccharides units covalently bound to each other.
  • Polysaccharides include starch, hyaluronic acid, amylose, amylopectin, dextran, cyclodextrin and glycogen.
  • aminosaccharide refers to a carbohydrate molecule where one or more hydroxy groups are replaced by an amino group. This includes the monosaccharides glucosamine and muramic acid and the polysaccharide chitin. The amino groups may be acetylated to include N-acetyl-D-glucosamine and N-acetyl-D-muramic acid.
  • deoxysaccharide refers to a carbohydrate molecule where one or more hydroxy groups are replaced by hydrogen. These include, for example, L-rhamnose (6-deoxy-L-mannose), L-fucose (6-deoxy-L-galactose) and D-fucose (rhodeose).
  • treatment refers to any treatment of a pathologic condition in a mammal, particularly a human, and includes: (i) preventing the pathologic condition from occurring in a subject which may be predisposed to the condition but has not yet been diagnosed with the condition and, accordingly, the treatment constitutes prophylactic treatment for the disease condition; (ii) inhibiting the pathologic condition, i.e., arresting its development; (iii) relieving the pathologic condition, i.e., causing regression of the pathologic condition; or (iv) relieving the conditions mediated by the pathologic condition.
  • therapeutically effective amount refers to that amount of a compound of the invention that is sufficient to effect treatment, as defined above, when administered to a mammal in need of such treatment.
  • the therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • salts includes, but is not limited to, salts well known to those skilled in the art, for example, mono-salts (e.g. alkali metal and ammonium salts) and poly salts (e.g. di- or tri-salts,) of the compounds of the invention.
  • Pharmaceutically acceptable salts of compounds of formula I are where, for example, an exchangeable group, such as hydrogen in -OH or -NH- is replaced with a pharmaceutically acceptable cation (e.g. a sodium, potassium, or ammonium ion) and can be conveniently be prepared from a corresponding compound of formula I by, for example, reaction with a suitable base.
  • salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, ⁇ -ketoglutarate, and ⁇ -glycerophosphate.
  • Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
  • salts may be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • a sufficiently basic compound such as an amine
  • a suitable acid affording a physiologically acceptable anion.
  • Alkali metal for example, sodium, potassium or lithium
  • alkaline earth metal for example, calcium
  • disease means any disease or other deleterious condition or disease in which therapeutic administration of a diuretic drug, or pharmaceutical composition, is known to play a role in treatment thereof.
  • diseases or conditions include, without limitation, hypertension, edema, acute renal failure, congestive heart failure, chronic renal failure, ascites, intra-ocular pressure or nephrotic syndrome and complications due to or exacerbated by those conditions.
  • diuretic means a drug or other substance tending to promote the formation and excretion of urine.
  • hypertension refers to a disorder characterized by elevated blood pressure.
  • Antibody refers to a member of a family of glycosylated proteins called immunoglobulins, which can specifically combine with an antigen.
  • Antigen refers to a compound which will give rise to antibody formation.
  • Antigenic determinant or “antigenic determinant site” refers to the actual site of antibody recognition of the antigen. The term is used interchangeably with “epitope”.
  • Carrier refers to a high molecular weight (macromolecular) polymeric material, usually a protein, to which an antigen or hapten can be bound or conjugated so as to facilitate antibody formation. Carriers can incorporate labels in their structure, if desired.
  • Conjugate refers to an antigen or hapten chemically bonded to a carrier; a conjugate can contain other groups, as well.
  • ELISA refers to an enzyme-linked immunosorbent assay which employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of antigen or antibody present in a sample.
  • a description of the ELISA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical Immunology by D. P. Sites et al, published by Lange Medical Publications of Los Altos, Calif., in 1982 , which is incorporated herein by reference.
  • EMIT refers to an enzyme-multiplied immunoassay technique which uses (1) an enzyme-labeled hapten, (2) specific antibody to the hapten, (3) pretreatment reagent, (4) buffered-enzyme substrate, and (5) standards to detect the amount of an unknown in a sample.
  • a description of the EMIT technique is found in Enzyme Immunoassay, edited by E. T. Maggio, published in 1980 by CRC Press, Inc., Boca Raton, Fla., particularly on pp. 141-150, 234-5, and 242-3 . These materials are incorporated by reference.
  • Epitope refers to that portion of a molecule which is specifically recognized by an antibody. It is also referred to as a determinant.
  • Fluoroimmunoassay refers to an antibody-based assay in which the species to be measured binds to, displaces or competes for binding with a material labelled with a fluorescent species in an antibody-ligand complex.
  • the complex is separated and the presence or absence of fluorescent species gives a measure of the amount of measured species.
  • the complex has different fluorescent properties than the uncomplexed fluorescent species so that the formation of the complex can be detected without separation of the complex.
  • fluoroimmunoassay techniques is found in " A Review of Fluoroimmunoassay and Immunofluorometric Assay", D. S. Smith et al. (1981) Ann. Clin. Biochem. (1981) 18:253-274 which is incorporated herein by reference.
  • Hapten refers to a compound, usually of low molecular weight, which when bound to a larger molecule can give rise to antibody formation.
  • Label refers to a detectable group in a molecule.
  • common labels are radioactive species useful in radioimmunoassays, fluorescent species useful in fluoroimmunoassays, and enzymatic species useful in the ELISA and EMIT methods and the like.
  • Ligand refers to any molecule which has an antibody combining site and can bind to a receptor.
  • Standard refers to a sample of a specific molecule present in a known concentration used to quantitate the same specific molecule in an unknown concentration of a different sample.
  • structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by a deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of this invention.
  • compositions comprising compounds of formula I.
  • the composition comprises compounds of formula I wherein R 1 , R 3 , R 5 , R 6 and R 7 are independently H, halogen or lower alkyl.
  • X 1 R 4 is -OC(O)CH 3 .
  • R 2 is -C(O)OR where R is preferably H or optionally substituted alkyl where alkyl may be methyl, ethyl, butyl, octyl or undecyl.
  • R 2 is -C(O)NHR where R is preferably H or optionally substituted alkyl, cycolalkyl, heterocycloalkyl, aryl or heteroaryl.
  • X 2 is -O- and R 8 is an optionally substituted saccharide, preferably a monosaccharide selected from an aldohexopyranose, aldopentopyranose, aldopentofuranose or ketose.
  • R 8 is D-galactose, D-mannose, D-ribose, D-fucose or L-rhamnose.
  • R 8 is D-glucose.
  • the compound of formula I is xanthurenic acid 8-O- ⁇ -D-glucoside.
  • X 2 is -O- and R 8 is -CH 2 CO 2 R or -CH 2 C(O)NHR where R is preferably H or optionally substituted alkyl, cycolalkyl, heterocycloalkyl, aryl or heteroaryl.
  • the pharmaceutical composition comprises a compound of formula I where X 2 R 8 is an acyl, phosphate, phosphonic acid, alkyl phosphonate or a sulfate group.
  • the compound is xanthurenic acid 8-O-sulfate.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound represented by formula I in combination one or more additional diuretic compounds or cardiovascular agents; and a pharmaceutically acceptable carrier.
  • compositions described herein are useful for treatment or prevention of hypertension, edema, acute renal failure, congestive heart failure, chronic renal failure, ascites, intra-ocular pressure or nephrotic syndrome and complications due to or exacerbated by those conditions.
  • additional therapeutic agents which are normally administered to treat or prevent that condition, may be administered together with the compounds of this invention.
  • additional diuretic agents which are normally administered to treat or prevent that condition, may be administered together with the compounds of this invention.
  • the additional diuretic agent is selected from the group consisting of a loop diuretic, thiazide diuretic, potassium-sparing diuretic, carbonic anhydrase inhibitor and osmotic diuretic.
  • the cardiovascular agent is selected from the group consisting of an angiotensin converting enzyme inhibitor, angiotensin II receptor antagonist, beta-adrenergic blocker, calcium channel blocker, cholesterol altering drug, triglyceride lowering agent, c-reactive protein lowering agent, homocysteine lowering agent, aspirin and its derivatives, ionotropic agent, antiarrhythmic agent and blood thinner (anticoagulant).
  • agents include, without limitation, furosemide, bumetanide, torsemide, ethacrynic acid, chlorothiazide, hydrochlorothiazide, spironolactone, amiloride, triamterene, acetazolamide, methazolamide, dichlorphenamide, hydroflumethiazide, methyclothiazide, indapamide, metolazone, polythiazide, chlorthalidone, dorzolamide, brinzolamide, glycerol, mannose, urea, lisinopril, moexipril, enalapril, irbesartan, valsartan, losartan, nadolol, propranolol, atenolol, timolol and bisoprolol.
  • the compounds of formula I can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms adapted to a selected route of administration, i.e., by oral, parenteral, intravenous, intramuscular, topical, or subcutaneous routes.
  • a mammalian host such as a human patient
  • the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
  • the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form.
  • the amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
  • the tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and devices.
  • the active compound may also be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, buffers or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
  • Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
  • Examples of useful dermatological compositions which can be used to deliver the compounds of the invention to the skin are disclosed in Jacquet et al. (U.S. Pat. No. 4,608,392 ), Geria (U.S. Pat. No. 4,992,478 ), Smith et al. (U.S. Pat. No. 4,559,157 ) and Wortzman (U.S. Pat. No. 4,820,508 ).
  • compositions may also be prepared in suitable forms for absorption through the mucous membranes of the nose and throat or bronchial tissues and may conveniently take the form of powder or liquid sprays or inhalants, lozenges, throat paints, etc.
  • the preparations may be presented as individual capsules, in liquid or semi-solid form, or may be used as drops, etc.
  • Topical applications may be formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints, powders, etc.
  • composition may, for example, be formulated as an intra-mammary preparation in either long acting or quick-release bases.
  • Useful dosages of the compounds of the invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949 .
  • the concentration of the compound(s) of the invention in a liquid composition will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%.
  • concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
  • compositions per unit dosage may contain from 0.1% to 99% of active material (compound I or salts thereof), the preferred range being from about 10-60%.
  • the composition will generally contain from about 15 mg to about 1,500 mg by weight of active ingredient based upon the total weight of the composition; however, in general, it is preferable to employ a dosage amount in the range of from about 250 mg to 1,000 mg.
  • the unit dosage is usually the pure compound in a slightly acidified sterile water solution or in the form of a soluble powder intended for solution.
  • Single dosages for injection, infusion or ingestion may be administered, i.e., 1-3 times daily, to yield levels of about 0.5-50 mg/kg, for adults.
  • the present disclosure further includes methods for the production of antibodies capable of specifically recognizing xanthurenic acid-8-O- ⁇ -D-glucoside.
  • Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • Such antibodies may be used, for example, in the detection of xanthurenic acid-8-O- ⁇ -D-glucoside in a biological sample, or, alternatively, as a method for the inhibition of abnormal xanthurenic acid-8-O- ⁇ -D-glucoside activity.
  • Such antibodies may be utilized as part of disease treatment methods, and/or may be used as part of diagnostic techniques whereby patients may be tested for abnormal levels of xanthurenic acid-8-O- ⁇ -D-glucoside.
  • conjugates of xanthurenic acid-8-O- ⁇ -D-glucoside with, for example, a carrier protein such as KLH or ovalbumin may be generated by activation of the xanthurenic acid-8-O- ⁇ -D-glucoside carboxyl group with, for example, a water soluble carbodiimide, such as EDC, to form a xanthurenic acid-8-O- ⁇ -D-glucoside bioconjugate immunogen.
  • Various host animals may be immunized by injection with the xanthurenic acid-8-O- ⁇ -D-glucoside bioconjugate immunogen in, for example, an adjuvanted protocol.
  • Such host animals include rabbits, mice, rats, goats and chickens, and the like.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG ( bacille Calmette-Guerin ) and Corynebacterium parvum.
  • BCG bacille Calmette-Guerin
  • Corynebacterium parvum bacille Calmette-Guerin
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as xanthurenic acid-8-O- ⁇ -D-glucoside bioconjugate immunogen.
  • an antigen such as xanthurenic acid-8-O- ⁇ -D-glucoside bioconjugate immunogen.
  • host animals such as those described above, may be immunized by injection with xanthurenic acid-8-O- ⁇ -D-glucoside bioconjugate immunogen supplemented with adjuvants as also described above.
  • xanthurenic acid-8-O- ⁇ -D-glucoside bioconjugate immunogens is introduced into a mammalian or avian host.
  • Suitable hosts include, for example, monkeys, cattle, rabbits, rats, mice, and the like. This is usually accomplished by subcutaneous injection as a solution in saline which has been emulsified with, for example, complete Freund's adjuvant. Animal antibody titers may be followed by ELISA.
  • a boost of xanthurenic acid-8-O- ⁇ -D-glucoside bioconjugate immunogen in, for example, Freund's incomplete adjuvant may serve to increase the antibody titer.
  • the antibodies are collected by bleeding the animal after about a month. The whole blood is allowed to clot at 25.degree. C. for several hours. Aqueous ammonium sulfate solution is added to achieve 40% by weight of aqueous solution, and the IgG fraction precipitates. The precipitate is collected by centrifugation and resuspended in saline or buffer solution to the desired concentration.
  • the purified antibody fraction may be further modified for use in diagnostic assay systems. Such modification may encompass linkage with enzymes such as lipozyme, lactoperoxidase, alkaline phosphatase and others for use in ELISA assays.
  • the antibody may be modified with fluorescent moieties. Optimally, this fluorescence may be quenched or enhanced upon binding of the antibody and antigen.
  • xanthurenic acid-8-O- ⁇ -D-glucoside, xanthurenic acid-8-O- ⁇ -D-glucoside conjugates and antibodies of this disclosure are also useful in the detection and diagnosis of various sodium imbalance disorders, particularly in providing high purity materials useful for calibration solutions for assay techniques, such as ELISA or EMIT.
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Köhler and Milstein, Nature, 256:495-7 (1975 ); and U.S. Patent No. 4,376,110 ), the human B-cell hybridoma technique ( Kosbor et al., Immunology Today, 4:72 (1983 ); Cote et al., Proc. Natl. Acad. Sci. USA, 80:2026-30 (1983 )), and the EBV-hybridoma technique ( Cole et al., in Monoclonal Antibodies And Cancer Therapy, Alan R.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAb of this disclosure may be cultivated in vitro or in vivo . Production of high titers of mAbs in vivo makes this the presently preferred method of production.
  • chimeric antibodies Morrison et al., Proc. Natl. Acad. Sci., 81:6851-6855 (1984 ); Takeda et al., Nature, 314:452-54 (1985 )) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • Single chain antibodies are typically formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Antibody fragments that recognize specific epitopes may be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed ( Huse et al., Science, 246:1275-81 (1989 )) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • the enzymes for use in the diagnostic reagents, standards or kits can vary widely, depending on the ease of conjugation, turnover rate, and the physiological fluid in which the unknown (analyte) is to be measured.
  • Representative enzymes of choice include hydrolysases, nucleases, amidases, esterases and the like which are found in U.S. Pat. No. 3,817,837 , which is incorporated herein by reference.
  • the present invention further includes the quantitation of xanthurenic acid-8-O- ⁇ -D-glucoside in human fluids (whole blood, urine, csf, serum, plasma, ocular, feces, sweat) which may be used to correlate to a disorder of sodium imbalance.
  • disorders may include hypertension (low renin, low angiotensin), congestive heart failure (edema), nephrotic syndrome, cirrosis of the liver, premenstrual edema, cyclical edema, and cardiogenic shock.
  • xanthurenic acid-8-O- ⁇ -D-glucoside quantitation may prove useful in postoperative settings, and battlefield settings, where the patient is given too much fluid; and potassium-sparing in conjunction with furosemide administration.
  • Xanthurenic acid-8-O- ⁇ -D-glucoside quantitation may also be useful in "Escape Syndrome” where a tumor inappropriately increases, affecting aldosterone; then sodium excretion decreases causing increase of fluid retention by two liters in the cardiovascular system. At this point, "escape” occurs by causing fluid and sodium loss and thus maintaining an overloaded state of two liters. It is proposed that the overloaded fluid state causes an increase in xanthurenic acid-8-O- ⁇ -D-glucoside, which in turn causes "escape” by stimulating sodium excretion and concomitant fluid loss.
  • a variety of methods may be employed to diagnose disease conditions associated with an excess or a deficiency of xanthurenic acid-8-O- ⁇ -D-glucoside.
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one specific antibody reagent described herein, which may be conveniently used, e.g ., in clinical settings, to diagnose patients exhibiting disease symptoms or at risk for developing disease.
  • Any human biological sample or tissue may be utilized in the diagnostics described below.
  • whole blood, urine, saliva, cerebral spinal fluid, serum, plasma, ocular, feces or sweat may be utilized.
  • Diagnostic methods for the detection of xanthurenic acid-8-O- ⁇ -D-glucoside in biological samples may involve, for example, immunoassays such as heterogeneous enzyme immunoassays which include solid phase enzyme-linked immunosorbent assays (ELISA); and solution phase homogeneous enzyme immunoassays which include enzyme-multiplied immunoassay technique (EMIT).
  • Xanthurenic acid-8-O- ⁇ -D-glucoside may be measured by high pressure liquid chomatography (HPLC), capillary electrophoresis (CE), or capillary electrophoresis-mass spectrometry (CE-MS) with optional pretreatment of, for example, a urine sample by solid phase extraction.
  • HPLC high pressure liquid chomatography
  • CE capillary electrophoresis
  • CE-MS capillary electrophoresis-mass spectrometry
  • SPR surface plasmon resonance
  • Immunoassays for xanthurenic acid-8-O- ⁇ -D-glucoside typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells, or cells that have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying xanthurenic acid-8-O- ⁇ -D-glucoside, and detecting the bound antibody by any of a number of techniques well known in the art.
  • the biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins.
  • a solid phase support or carrier such as nitrocellulose, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins.
  • the support may then be washed with suitable buffers followed by treatment with the detectably labeled gene-specific antibody.
  • the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on solid support may then be detected by conventional means.
  • solid phase support or carrier are intended to encompass any support capable of binding an antigen or an antibody.
  • Well-kno.wn supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present disclosure.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • the anchored component may be immobilized by non-covalent or covalent attachments.
  • Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of a xanthurenic acid-8-O- ⁇ -D-glucoside bioconjugate and drying.
  • an immobilized antibody preferably a monoclonal antibody, specific for xanthurenic acid-8-O- ⁇ -D-glucoside may be used to anchor the protein to the solid surface.
  • the surfaces may be prepared in advance and stored. In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component.
  • any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g ., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
  • competitive inhibition assays are often used to measure small analytes, such as xanthurenic acid-8-O- ⁇ -D-glucoside, because competitive inhibition assays only require the binding of one antibody rather than two as is used in standard ELISA formats. Because of the probability for steric hindrance occurring when two antibodies attempt to bind to a small molecule at the same time, a sandwich assay format may not be feasible, therefore a competitive inhibition assay may be preferable.
  • the sample and conjugated analyte are added in steps like a sandwich assay, while in a classic competitive inhibition assay, these reagents are incubated together at the same time.
  • a monoclonal antibody (MAb) is coated onto a 96-well microtiter plate.
  • the MAb captures free analyte out of the sample.
  • a known amount of analyte labeled with either biotin or HRP is added.
  • the labeled analyte will then also attempt to bind to the MAb adsorbed onto the plate, however, the labeled analyte is inhibited from binding to the MAb by the presence of previously bound analyte from the sample.
  • the labeled analyte will not be bound by the monoclonal on the plate if the monoclonal has already bound unlabeled analyte from the sample.
  • the amount of unlabeled analyte in the sample is inversely proportional to the signal generated by the labeled analyte. The lower the signal, the more unlabeled analyte there is in the sample.
  • a standard curve can be constructed using serial dilutions of an unlabeled analyte standard. Subsequent sample values can then be read off the standard curve as is done in the sandwich ELISA formats.
  • the classic competitive inhibition assay format requires the simultaneous addition of labeled (conjugated analyte) and unlabeled analyte (from the sample). Both labeled and unlabeled analyte then compete simultaneously for the binding site on the monoclonal capture antibody on the plate. Like the sequential competitive inhibition format, the colored signal is inversely proportional to the concentration of unlabeled target analyte in the sample.
  • Detection of labeled analyte may be made by using a peroxidase substrate such as TMB, which can be read on a microtiter plate reader.
  • a peroxidase substrate such as TMB
  • a 96-well microtiter plate format can test 21 samples in triplicate and 37 samples in duplicate.
  • antibodies, or fragments of antibodies useful in the present disclosure may be used to quantitatively or qualitatively detect the presence of xanthurenic acid-8-O- ⁇ -D-glucoside. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection.
  • the antibodies (or fragments thereof) useful in the present disclosure may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of xanthurenic acid-8-O- ⁇ -D-glucoside.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present disclosure.
  • the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • EIA enzyme immunoassay
  • EIA enzyme immunoassay
  • the enzyme that is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • Enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986 ).
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • fluorescent labeling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series, These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediamine-tetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediamine-tetraacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • EMIT Enzyme-multiplied immunoassay techniques
  • Various EMIT methods could be used in both qualitative and quantitative assays by, for example, the methods described by Dias, et al. (see Dias et al., "The EMIT Cyclosporine Assay: development of application protocols for the Boehringer Mannheim Hitachi 911 and 917 analyzers" Clin. Biochem. 1997 Mar;30(2):155-62 ).
  • EMIT in the clinical setting include (1) minimal or no sample preparation, (2) small sample size, (3) excellent correlation with other methods such as HPLC and RIA, (4) rapid time since there is no need for separation of free and bound enzyme labels (less than one minute), ease of adaptation to most general chemistry analyzers.
  • SPR Surface plasmon resonance
  • McDonnell, J.M. "Surface plasmon resonance: towards an understanding of the mechanisms of biological molecular recognition. Current Opinion in Chemical Biology, 2001. 5(5): p. 572-577 ; Rich, R.L., et al., "High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using BIACORE” Analytical Biochemistry, 2001.
  • Various flow-through cells may be constructed and placed in-line with commercially available SPR biosensing instruments, such as those with trademark Biocore.
  • the specific antibody may be immobilized on an SPR-active gold-coated glass slide which forms one wall of a flow-cell; and the analyte xanthurenic acid-8-O- ⁇ -D-glucoside in an aqueous buffer solution is injected to flow across the flow-cell.
  • the optical reflectivity of the gold changes very sensitively with the presence of biomolecules on the gold suface or in a thin coating on the gold.
  • the extent of binding between the solution-phase interactant and the immobilized antibody is easily observed and quantified by monitoring this reflectivity change.
  • High pressure liquid chromatography may be employed to detect xanthurenic acid-8-O-b-D-glucoside in urine.
  • urine sample solid phase extraction on a C 18 cartridge and subsequent passage over a cation-exchange resin column may be employed.
  • Sample detection may be via UV, fluorescence, or light scattering techniques, (see, for example, Marsilio et al., Clinical Chemistry. 1998;44:1685-1691 .)
  • Capillary electrophoresis (CE), or capillary electrophoresis-mass spectrometry (CE-MS) with optional pretreatment of urine sample by solid phase extraction may also be employed to quantitate xanthurenic acid-8-O- ⁇ -D-glucoside in biological samples.
  • Step 1 Synthesis of xanthurenic acid 2, 3, 4, 6-tetra-O-acetyl 8-O- ⁇ -D-glucoside. (See, for example, Real, et al ., J. Biol. Chem., 1990, 265(13), 7407-7412)
  • Xanthurenic acid was obtained commercially from Aldrich, Milwaukee, WI. Xanthurenic acid (930 mg, 4.53 mmol) in aqueous 1M NaOH (10 mL) was cooled to 10°C. 2, 3, 4, 6-tetra-O-acetyl8- ⁇ -D-glucopyranosylbromide (2.03 g, 4.94 mmol) in acetone (16 mL) was added dropwise over 10 minutes. The solution was allowed to warm to room temperature over 4 hours. Additional aqueous 1M NaOH (3 mL) was added slowly over 30 minutes and solution stirred 30 minutes. The mixture was extracted with water and diethyl ether.
  • the aqueous portion was acidified to pH 3.5 and further extracted with 1:1 tetrahydrofuran/ethylacetate. The combined organic layers were washed with saturated aqueous NaCl and dried over magnesium sulfate. Following filtration, the crude xanthurenic acid tetra-O-acetyl 8- ⁇ -D-glucoside intermediate was concentrated in vacuo to give approximately 1 gram of a crude residue. The residue was triturated with 4:1 dimethylsulfoxide/water (28 mL), filtered and dried to return 215 mg of xanthurenic acid tetra-O-acetyl 8- ⁇ -D-glucoside as an off-white solid intermediate.
  • Step 2 Xanthurenic acid 8-O- ⁇ -D-glucoside.
  • Xanthurenic acid 2,3,4,6-tetra-O-acetyl 8-O- ⁇ -D-glucoside from step 1 (190 mg, 0.35 mmol) was added to a solution of 95% sodium methoxide (40 mg, 0.7 mmol) in methanol (5 mL). The mixture was stirred for one hour. The mixture was adjusted to pH 3.5 with aqueous 1M HCl. The slurry was diluted with 20 mL diethyl ether and filtered. The filter cake was washed with 1:1 methanol/diethyl ether and dried in vacuo to give 118 mg of xanthurenic acid 8-O- ⁇ -D-glucoside in an 82% yield.
  • Example 3 Isolation of xanthurenic acid 8-O-sulfate and xanthurenic acid 8-O- ⁇ -D-glucoside.
  • Urine samples from human uremic patients were collected over 24-hour periods. The typical collection volume was of 2-3 liters per patient. Individual samples were lyophilized to dryness and reconstituted with 100 mL of deionized water. The reconstituted samples (25 mL load volume) were size-fractionated using gel filtration Sephadex G-25 column chomatography with elution by 10 mM ammonium acetate, pH 6.8 at 10°C and monitored by UV at 285 nm and conductivity. Later eluting (post-salt peak) 10 mL fractions with UV activity and osmolality ⁇ 100 mOsm were screened for biological activity with the frog skin assay of Bricker et al. (Kidney International Vol 44 (1993) Nov; 44 (5): 937-47 ). Fractions with activity (10 mL each) were concentrated by lyophilization and reconstituted with 1 mL dionized water.
  • Fractions containing either the xanthurenic acid 8-O- ⁇ -D-glucoside or the xanthurenic acid 8-O-sulfate were separately pooled and concentrated on a Savant Speed-Vac Plus (SC210A) with medium heat.
  • the pools were resubjected to isocratic HPLC on an analytical scale using a reverse phase C-18 HPLC column: Phenomenex P/NO 00G-4375-E0, SYNERGI 4u Hydro-RP 80A 250 x 4.6 mm, 4 micron.
  • the HPLC purification was run in isocratic mode at 1 mL/minute with 8% methanol and 0.1% 0.1M pyridium acetate at 35°C.
  • Xanthurenic acid 8-O- ⁇ -D-glucoside formed crystals upon concentration by this technique.
  • xanthurenic acid 8-O- ⁇ -D-glucoside C 16 H 17 NO 9
  • ESI-MS m/z 367.09, 368.094 (M+H) and 229 (M -glucose + Na + );
  • 1 H-NMR 500 MHz, D 2 O
  • Example 4 Natriuretic response to synthetic xanthurenic acid 8-O- ⁇ -D-glucoside (2 ⁇ g i.v.) in a normal Sprague Dawley rat.
  • a female Sprague Dawley rat 250 g was anesthetized lightly with ether and a tail vein catheter was placed using PE10 tubing. Additionally, a urethra catheter was inserted using KY jelly and 2% lidocaine as a lubricant. The rat was restrained in a modified Plexiglas tube so that urine could be collected in 1.5-mL microcentrifuge tubes. Saline infusion started at time zero at 0.02 mL/min for the duration of the assay. The same i.v. catheter was used to inject the test compound.
  • Synthetic xanthurenic acid 8-O- ⁇ -D-glucoside 2 ⁇ g was injected at the time indicated in a 1-mL volume in saline over the course of 10 minutes. The tubes were centrifuged at 14,000 rpm to separate any RBC's from the urine. Na + and K + concentrations in the urine were measured with respective ion selective electrodes. The Na + and K + excretion rates were calculated by: (vol of urine / time of collection period) x (ion urine concentration). Results are shown in Figures 1-4 . Synthetic xanthurenic acid 8-O- ⁇ -D-glucoside at 2 ug i.v. caused a sustained natriuretic response in a normal rat.
  • Na + excretion was due more to increased Na + urine concentration than increased urine volume as shown in Figures 3 and 4 .
  • concentration of the test compound was 10 -6 M, a possible minimum dose.
  • synthetic, underivatized xanthurenic acid at 2 ug i.v. did not increase Na + excretion.
  • subsequent administration of synthetic xanthurenic acid 8-O- ⁇ -D-glucoside did cause natriuresis.
  • Example 5 Natriuretic response to synthetic xanthurenic acid 8-O- ⁇ -D-glucoside (10 ⁇ g i.v.) in a normal Sprague Dawley rat.
  • a female Sprague Dawley rat 250 g was anesthetized lightly with ether and a tail vein catheter was placed using PE10 tubing. Additionally, a urethra catheter was inserted using KY jelly and 2% lidocaine as a lubricant. The rat was restrained in a modified Plexiglas tube so that urine could be collected in 1.5-mL microcentrifuge tubes. Saline infusion started at time zero at 0.02 mL/min for the length of the assay. The same i.v. catheter was used to inject the test compound. Synthetic xanthurenic acid 8-O- ⁇ -D-glucoside, 10 ⁇ g i.v.
  • Urine production decreased 140 min after administration resulting in decreased natriuresis.
  • Na + excretion rate increased from 2 uEq/min to 10 uEq/min, seen in Figure 6 .
  • Example 6 Natriuretic response to synthetic xanthurenic acid 8-O-B-D-glucoside (10 ⁇ g) followed by furosemide (100 ⁇ g) in a normal Sprague Dawley Rat, (oral administration).
  • a female Sprague Dawley rat (250 g) was anesthetized lightly with ether and a urethra catheter was inserted using KY jelly and 2% lidocaine as a lubricant.
  • the rat was restrained in a modified Plexiglas tube so that urine could be collected in 1.5-mL microcentrifuge tubes. No saline infusion was administered.
  • Synthetic xanthurenic acid 8-O- ⁇ -D-glucoside was injected with a feeding needle at the time indicated in a 1-mL volume of water over the course of 1 minute. Sixty minutes later 100 ⁇ g of furosemide was similarly administered with a feeding needle.
  • Example 7 Natriuretic response to isolated xanthurenic acid 8-O-B-D-glucoside (3 ⁇ g) followed by furosemide (20 ⁇ g) in uremic Sprague Dawley rat (i.v.).
  • Sprague-Dawley rat Female Sprague-Dawley rat, weighing 225 g, was made uremic by tying off one kidney and 30-50% of the second kidney. Two weeks later the rat was ready to test for natriuretic activity.
  • the rat was anesthetized lightly with ether and a tail vein catheter was placed using PE10 tubing. Additionally, a urethra catheter was inserted using KY jelly and 2% lidocaine as a lubricant, The rat was restrained in a modified Plexiglas tube so that urine could be collected in 1.5-mL microcentrifuge tubes. Saline infusion started at time zero at 0.02 mL/min for the length of the assay. The same i.v.
  • Results are shown in Figures 13-16 .
  • Isolated xanthurenic acid 8-O- ⁇ -D-glucoside at 3 ⁇ g i.v. caused a sustained natriuretic response in the uremic rat.
  • the time course of Na + excretion peaked at 70 minutes in the uremic rat shown in Figure 13 , which is a similar time course to the synthetic xanthurenic acid 8-O- ⁇ -glucoside in the normal rat shown in Figure 1 .
  • K + excretion in Figure 14 and 15 did not increase in response to isolated xanthurenic acid 8-0- ⁇ -glucoside.
  • the diuretic response to furosemide was classic in terms of its time course as well as the Na + excretion.
  • furosemide Normally, furosemide also causes K + excretion to the extent that K + supplementation is necessary in the clinical use of furosemide.
  • a similar effect in nonnal rat is illustrated in Figure 25 (furosemide only).
  • Pretreatment with xanthurenic acid 8-O- ⁇ -glucoside followed by furosemide prevented the typical increase in K + excretion in this assay shown in Figures 14 and 15 .
  • Example 8 Na + and K + urine excretion response to isolated xanthurenic acid 8-O-sulfate (2.0 ⁇ g) in uremic Sprague Dawley rat (i.v. administration).
  • the rat was anesthetized lightly with ether and a tail vein catheter was placed using PE10 tubing. Additionally, a urethra catheter was inserted using KY jelly and 2% lidocaine as a lubricant.
  • the rat was restrained in a modified Plexiglas tube so that urine could be collected in 1.5-mL microcentrifuge tubes. Saline infusion started at time zero at 0.02 mL/min for the length of the assay. The same i.v. catheter was used to inject the test compound.
  • Isolated xanthurenic acid 8-O-sulfate 2 ⁇ g was injected at the time indicated in a 1 mL volume in saline over the course of 10 minutes. Then saline infusion was returned to 0.02 mL/min for the duration of the assay.
  • the tubes were centrifuged at 14,000 rpm to separate any RBC's from the urine. Na + and K + concentrations in the urine were measured with respective ion selective electrodes. The Na + and K + excretion rates were calculated by: (vol of urine / time of collection period) x (ion urine concentration).
  • Results are shown in Figures 17-20 .
  • the time course of the natriuresis peaked within 30 minutes and then approached control levels within 70 minutes of treatment, seen in Figures 17 and 18 .
  • K + excretion did not increase in response to isolated xanthurenic acid 8-O-sulfate, shown in Figures 18 and 19 .
  • Example 9 Na + and K + urine excretion response to synthetic xanthurenic acid 8-0-sulfate (20 ⁇ g) in normal Sprague Dawley rat (oral administration).
  • a female Sprague Dawley rat (250 g) was anesthetized lightly with ether and a urethra catheter was inserted using KY jelly and 2% lidocaine as a lubricant.
  • the rat was restrained in a modified Plexiglas tube so that urine could be collected in 1.5-mL microcentrifuge tubes. No saline infusion was administered.
  • Xanthurenic acid 8-0-sulfate was injected with a feeding needle at the time indicated in a 1-mL volume of water over the course of 1 minute.
  • the tubes were centrifuged at 14,000 rpm to separate any RBC's from the urine. Na + and K + concentrations in the urine were measured with respective ion selective electrodes.
  • the Na + and K + excretion rates were calculated by: (vol of urine / time of collection period) x (ion urine concentration). Results are shown in Figures 21-24 .
  • Xanthurenic acid 8-0-sulfate (20 ug) was not orally active with respect to causing a natriuretic response in a normal rat.
  • the Na + urine concentration remained below 50 mM in Figure 21 whereas in i.v. administered xanthurenic acid 8-O-sulfate the Na + urine concentration increased from 60 mM to 160 mM in Figure 19 .
  • Xanthurenic acid 8-O- ⁇ -D-glucoside was orally active in a normal rat by increasing the Na + urine concentration to 160 mM in Figure 11 .
  • Example 10 Protocol for polyclonal antibody production against xanthurenic acid 8-O-beta-D-glucoside and xanthurenic acid 8-sulfate.
  • the following protocol describes the production of polyclonal antibodies to xanthurenic acid 8-O-beta-D-glucoside in the development of an ELISA assay to detect and measure xanthurenic acid 8-O-beta-D-glucoside in urine and plasma.
  • Xanthurenic acid 8-O-beta-D-glucoside is covalently linked to the large protein KLH to elicit an immune response in rabbits.
  • ASBA is used as a spacer arm (12 ⁇ ) between xanthurenic acid 8-O-beta-D-glucoside and KLH. The spacer arm allows better presentation of xanthurenic acid 8-O-beta-D-glucoside in the immune response.
  • xanthurenic acid 8-O-beta-D-glucoside is similarly linked to BSA which is then coated onto ELISA wells.
  • the rabbit serum is then screened for xanthurenic acid 8-O-beta-D-glucoside antibodies by incubating the rabbit serum with the BSA conjugated to xanthurenic acid 8-O-beta-D-glucoside.
  • These rabbit antibodies are detected by binding goat-anti-rabbit antibodies that are conjugated to the reporter enzyme horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • TMB tetramethylbenzidine
  • xanthurenic acid 8-O-beta-D-glucoside and xanthurenic acid 8-sulfate will be established by both ELISA and SPE/HPLC techniques.

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US8697674B2 (en) * 2004-12-29 2014-04-15 Naturon, Inc. Xanthurenic acid derivative pharmaceutical compositions and methods related thereto
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Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3817837A (en) 1971-05-14 1974-06-18 Syva Corp Enzyme amplification assay
GB2092130A (en) * 1980-07-28 1982-08-11 Hodogaya Chemical Co Ltd 4-Oximino-1,2,3,4- tetrahydroquinoline Derivatives
US4366241A (en) 1980-08-07 1982-12-28 Syva Company Concentrating zone method in heterogeneous immunoassays
US4376110A (en) 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies
US4559157A (en) 1983-04-21 1985-12-17 Creative Products Resource Associates, Ltd. Cosmetic applicator useful for skin moisturizing
US4608392A (en) 1983-08-30 1986-08-26 Societe Anonyme Dite: L'oreal Method for producing a non greasy protective and emollient film on the skin
US4820508A (en) 1987-06-23 1989-04-11 Neutrogena Corporation Skin protective composition
US4938949A (en) 1988-09-12 1990-07-03 University Of New York Treatment of damaged bone marrow and dosage units therefor
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US4992478A (en) 1988-04-04 1991-02-12 Warner-Lambert Company Antiinflammatory skin moisturizing composition and method of preparing same
US5124152A (en) * 1991-01-07 1992-06-23 Fisons Corporation Parenteral formulation of metolazone

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1334705A (en) 1970-06-19 1973-10-24 Ici Ltd Pharmaceutical compositions containing kynurenic acid derivatives
ZA801575B (en) * 1979-03-27 1981-03-25 Boots Co Ltd Therapeutic agents
JPS55151511A (en) 1979-05-16 1980-11-26 Sankyo Co Ltd Antiallergic agent consisting mainly of kynurenic ester derivative
DE2939304A1 (de) * 1979-09-28 1981-04-16 Wella Ag Mittel und verfahren zur oxidativen faerbung von haaren
US4552884A (en) * 1984-01-13 1985-11-12 The Boots Company Plc Method of treating heart disease
US4656188A (en) * 1985-10-09 1987-04-07 Merck & Co., Inc. Ace inhibitors in macular degeneration
US5106630A (en) * 1988-07-11 1992-04-21 Neal S. Bricker Natriuretic hormone
CA2134611C (en) * 1994-10-28 2002-12-24 Richard John Yarwood Process for preparing solid pharmaceutical dosage forms
JP2004506915A (ja) 2000-08-18 2004-03-04 ザ　ゼネラル　ホスピタル　コーポレーション 白内障を阻害又は促進する物質を同定する方法及びその使用
FR2827045B1 (fr) 2001-07-05 2007-08-10 Univ Pasteur Methodes et compositions pour la selection et le developpement de nouveaux agents pharmacologiques ou de nouveaux medicaments
US20030152622A1 (en) * 2001-10-25 2003-08-14 Jenny Louie-Helm Formulation of an erodible, gastric retentive oral diuretic
AU2002950217A0 (en) 2002-07-16 2002-09-12 Prana Biotechnology Limited 8- Hydroxy Quinoline Derivatives
US8697674B2 (en) * 2004-12-29 2014-04-15 Naturon, Inc. Xanthurenic acid derivative pharmaceutical compositions and methods related thereto
US20070049559A1 (en) 2005-06-28 2007-03-01 Bruce Pfeffer Preparations comprising arylazine substituted with a carbonylic moiety to increase the activity of Gelatinase A in ocular cells

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3817837A (en) 1971-05-14 1974-06-18 Syva Corp Enzyme amplification assay
GB2092130A (en) * 1980-07-28 1982-08-11 Hodogaya Chemical Co Ltd 4-Oximino-1,2,3,4- tetrahydroquinoline Derivatives
US4376110A (en) 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies
US4366241A (en) 1980-08-07 1982-12-28 Syva Company Concentrating zone method in heterogeneous immunoassays
US4366241B1 (zh) 1980-08-07 1988-10-18
US4559157A (en) 1983-04-21 1985-12-17 Creative Products Resource Associates, Ltd. Cosmetic applicator useful for skin moisturizing
US4608392A (en) 1983-08-30 1986-08-26 Societe Anonyme Dite: L'oreal Method for producing a non greasy protective and emollient film on the skin
US4820508A (en) 1987-06-23 1989-04-11 Neutrogena Corporation Skin protective composition
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US4992478A (en) 1988-04-04 1991-02-12 Warner-Lambert Company Antiinflammatory skin moisturizing composition and method of preparing same
US4938949A (en) 1988-09-12 1990-07-03 University Of New York Treatment of damaged bone marrow and dosage units therefor
US5124152A (en) * 1991-01-07 1992-06-23 Fisons Corporation Parenteral formulation of metolazone

Non-Patent Citations (34)

* Cited by examiner, † Cited by third party
Title
"Diagnostic Horizons", vol. 2, 1978, MICROBIOLOGICAL ASSOCIATES QUARTERLY, article "The Enzyme Linked Immunosorbent Assay (ELISA", pages: 1 - 7
BIRD, SCIENCE, vol. 242, 1988, pages 423 - 26
BRICKER ET AL., KIDNEY INTERNATIONAL, vol. 44, no. 5, November 1993 (1993-11-01), pages 937 - 47
BUTLER, METH. ENZYMOL., vol. 73, 1981, pages 482 - 523
C. D. CAIN ET AL: "Identification of xanthurenic acid 8-O-beta-D-glucoside and xanthurenic acid 8-O-sulfate as human natriuretic hormones", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 104, no. 45, 1 January 2007 (2007-01-01), pages 17873 - 17878, XP055018435, ISSN: 0027-8424, DOI: 10.1073/pnas.0705553104 *
COLE ET AL.: "Monoclonal Antibodies And Cancer Therapy", 1985, ALAN R. LISS, INC., pages: 77 - 96
COTE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 80, 1983, pages 2026 - 30
D. P. SITES ET AL.: "Basic and Clinical Immunology 4th Edition", 1982, LANGE MEDICAL PUBLICATIONS OF LOS ALTOS, article "Chapter 22"
D. S. SMITH ET AL.: "A Review of Fluoroimmunoassay and Immunofluorometric Assay", ANN. CLIN. BIOCHEM., vol. 18, 1981, pages 253 - 274, XP008037341
DIAS ET AL.: "The EMIT Cyclosporine Assay: development of application protocols for the Boehringer Mannheim Hitachi 911 and 917 analyzers", CLIN. BIOCHEM., vol. 30, no. 2, March 1997 (1997-03-01), pages 155 - 62
E. T. MAGGIO,: "Enzyme Immunoassay", 1980, CRC PRESS, INC., pages: 141 - 150,234-5,242-3
ETSUKO SHIRAO ET AL: "Identification of a Novel Fluorophore, Xanthurenic Acid 8- O -[beta]- -glucoside in Human Brunescent Cataract", EXPERIMENTAL EYE RESEARCH, vol. 73, no. 4, 1 October 2001 (2001-10-01), pages 421 - 431, XP055018440, ISSN: 0014-4835, DOI: 10.1006/exer.2001.1051 *
GOODMAN: "Gilman's The Pharmacological Basis for Therapeutics", 2001, MACGRAW-HILL, pages: 772
HE ET AL., J CHROMATOGR B BIOMED SCI APPL., vol. 727, no. 1-2, 30 April 1999 (1999-04-30), pages 43 - 52
HUSE ET AL., SCIENCE, vol. 246, 1989, pages 1275 - 81
HUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 83
ISHIKAWA ET AL.,: "Enzyme Immunoassay", 1981, IGAKU-SHOIN
JAMA, 21 May 2003 (2003-05-21), Retrieved from the Internet <URL:w w.nhlbi.nih.go>
KOHLER; MILSTEIN, NATURE, vol. 256, 1975, pages 495 - 7
KOSBOR ET AL., IMMUNOLOGY TODAY, vol. 4, 1983, pages 72
MAGGIO: "Enzyme Immunoassay", 1980, CRC PRESS
MARSILIO ET AL., CLINICAL CHEMISTRY, vol. 44, 1998, pages 1685 - 1691
MCDONNELL, J.M.: "Surface plasmon resonance: towards an understanding of the mechanisms of biological molecular recognition", CURRENT OPINION IN CHEMICAL BIOLOGY, vol. 5, no. 5, 2001, pages 572 - 577, XP002981400, DOI: doi:10.1016/S1367-5931(00)00251-9
MORRISON ET AL., PROC. NATL. ACAD. SCI., vol. 81, 1984, pages 6851 - 6855
REAL ET AL., J. BIOL. CHEM., vol. 265, no. 13, 1990, pages 7407 - 7412
RICH, R.L. ET AL.: "High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using BIACORE", ANALYTICAL BIOCHEMISTRY, vol. 296, no. 2, 2001, pages 197 - 207, XP002307383, DOI: doi:10.1006/abio.2001.5314
SATO T ET AL: "Studies on conjugation S35-sulfate with phenolic compounds", JOURNAL OF BIOCHEMI, OXFORD UNIVERSITY PRESS, GB, vol. 49, no. 2, 1 February 1961 (1961-02-01), pages 164 - 168, XP008119838, ISSN: 0021-924X *
SIMON R P ET AL: "Inhibition of excitatory neurotransmission with kynurenate reduces brain edema in neonatal anoxia", NEUROSCIENCE LETTERS, LIMERICK, IE, vol. 71, no. 3, 21 November 1986 (1986-11-21), pages 361 - 364, XP024375562, ISSN: 0304-3940, [retrieved on 19861121], DOI: 10.1016/0304-3940(86)90648-8 *
TAKEDA ET AL., NATURE, vol. 314, 1985, pages 452 - 54
THEODORIDIS ET AL., J CHROMATOGR B BIOMED SCI APPL., vol. 745, no. 1, 4 August 2000 (2000-08-04), pages 49 - 82
VOLLER ET AL., J. CLIN. PATHOL., vol. 31, 1978, pages 507 - 20
VOLLER, RIC CLIN LAB, vol. 8, 1978, pages 289 - 98
WARD ET AL., NATURE, vol. 334, 1989, pages 544 - 46
WEINTRAUB, B.: "Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques", March 1986, THE ENDOCRINE SOCIETY

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